Lectin anti-Glut1 Merge

Glu

t1 D

S m

uta

nt

Wild

-typ

e

a

25mm

0

2

4

6

8

Nu

mb

er

of

ve

ss

el

bra

nc

h p

oin

ts WT Mut N.S.

0

1 5

3 0

4 5

6 0

Fre

qu

en

cy

dis

trib

uti

on

(%

)

N.S.

N.S.

N.S. N.S. N.S. N.S.

WT Mut

b c

Supplementary Figure 1. Brain microvasculature of Glut1 DS and WT mice. (a) Immunohistochemistry of brain sections with labeled lectin or an antibody against the Glut1 protein demonstrates perfect alignment of the two probes. Depicted are representative sections of the thalamus from a 6-month old Glut1 DS mutant and WT littermate. A frequency distribution of individual capillaries of various sizes (lengths) (b) or an assessment of vessel branch points (c) fails to detect differences between the cerebral microvasculature of Glut1 DS mutants and WT littermates at 2 weeks. P > 0.05, t test, N ≥ 3 mice of each genotype.

0 .0 0 0

0 .0 0 1

0 .0 0 21 .0

1 .5

2 .0

0 .0

0 .2

0 .4

0 .6

0 .83 0

3 5

4 0

4 5

5 0

IgG

co

nc

en

tra

tio

n (

mg

/mL

)

Alb

um

in c

on

ce

ntra

tion

(mg

/mL

)

N.S.

N.S.

N.S.

N.S.

Serum Serum CSF CSF

WT Mut

a

Glu

t1 D

S m

uta

nt

Wild

-typ

e

Wild

-typ

e +

KA

Lectin Alexa-594-BSA b

Merge

25mm

Supplementary Figure 2. The integrity of the blood-brain barrier is preserved in Glut1 DS model mice. (a) CSF and serum concentrations of albumin and IgG remain unchanged in Glut1 DS model mice. P > 0.05, t test, N ≥ 4 mice of each genotype. (b) The BBB of mutant mice remains intact as determined by the absence of fluorescently labeled albumin (BSA) in the brain parenchyma following administration of the probe. Representative thalamic sections are depicted. Note fluorescence in the parenchyma of mice pre-treated with kainic acid.

WT

Mut

0 .0

0 .5

1 .0

1 .5

Re

lati

ve

RN

A e

xp

res

sio

n

(Ab

c2

g G

AP

DH

)

N.S.

WT

Mut

0 .0

0 .5

1 .0

1 .5

Re

lati

ve

RN

A e

xp

res

sio

n

(Ab

cb

1b G

AP

DH

) N.S.

WT

Mut

0 .0

0 .5

1 .0

1 .5

2 .0

Re

lati

ve

RN

A e

xp

res

sio

n

(Cd

h5 G

AP

DH

)

N.S.

b

WT

Mut

0 .0

0 .5

1 .0

1 .5

Re

lati

ve

RN

A e

xp

res

sio

n

(Plv

ap

/GA

PD

H) N.S.

e f g h

N.S.

WT

Mut

0 .0

0 .5

1 .0

1 .5R

ela

tiv

e R

NA

ex

pre

ss

ion

(Cld

n3 G

AP

DH

)

WT

Mut

0 .0

0 .5

1 .0

1 .5

Re

lati

ve

RN

A e

xp

res

sio

n

(Cld

n1

2 G

AP

DH

) N.S.

WT

Mut

0 .0

0 .5

1 .0

1 .5

Re

lati

ve

RN

A e

xp

res

sio

n

(Cld

n5 G

AP

DH

) N.S.

c d

Merge TMR-Biocytin Glut1

Glu

t1 D

S m

uta

nt

Wild

-typ

e

Wild

-typ

e +

KA

25mm

a

Supplementary Figure 3. The Glut1-deficient blood-brain barrier is impervious to small molecules and fails to exhibit perturbations in BBB signature genes. (a) Immunohistochemistry of sections from the thalamus of mice administered the small-molecule tracer TMR-bioctyin indicates that haploinsufficiency of Glut1 does not compromise the BBB. Note absence of major differences in the intensity of TMR-biocytin in the neuropil of wild-type and mutant mice. Faint TMR-biocytin puncta outside the vessels is considered normal and observed in unaffected mice too (Ref. 22). (b – h) Graphs depicting quantitative PCR results of the expression of BBB endothelial “signature” genes in brain tissue of 20-week old Glut1 DS model mice and WT controls. P > 0.05, t test, N ≥ 6 mice of each genotype.

0 2 0 4 0 6 0 8 0

0

5

1 0

1 5

2 0

M in u te s

OC

R (

pm

ole

s/m

in/m

g p

rote

in)

0 2 0 4 0 6 0 8 0

0 .0

0 .5

1 .0

1 .5

2 .0

2 .5

M in u te s

EC

AR

(m

pH

/min

/mg

pro

tein

)

Vehicle Oligomycin 2,4-DNP Rotenone

Vehicle Oligomycin 2,4-DNP Rotenone

WT

WT

Mut

Mut

b

a

Supplementary Figure 4. Glycolytic flux and maximal respiratory capacity declines in Glut1 DS cells. (a) Glycolytic flux, as assessed by the rate of extra-cellular acidification (ECAR), under basal conditions and following injection of metabolic drugs into the culture medium, is reduced in Glut1 DS cells. (b) Mitochondrial respiration, as assessed by oxygen consumption rate (OCR) depicts an inability of the mutant cells to attain the maximal respiratory capacity of wild-type cells – see traces following administration of 2, 4-Dinitrophenol (2, 4-DNP). For quantification of the parameters, refer to Fig. 3.

mGlut1 hGlut1 eGFP

anti

-Glu

t1

anti

-eG

FP

DA

PI

Mer

ge

a

15mm

anti

-eG

FP

Glut1 DS + eGFP Glut1 DS + mGlut1+ eGFP Glut1 DS + hGlut1+ eGFP WT + eGFP

b

20mm

Supplementary Figure 5. Analysis of Glut1 constructs in cultured cells. (a) Glut1 DS patient fibroblasts transfected with either a murine or human Glut1 cDNA construct but not an eGFP cassette express high levels of the Glut1 protein (arrows). (b) Efficient transduction of Glut1 DS fibroblasts subjected to the glucose uptake assay. Depicted are cells from each of the experiments stained for eGFP which was co-transfected into the fibroblasts along with the Glut1 constructs.

0

4 0 0

8 0 0

1 2 0 0

La

ten

cy

to

fa

ll (

se

c) ***

*** *** ***

N.S. N.S. N.S. N.S.

8 wks 12 wks 16 wks 20 wks

Mut + vehicle Mut + AAV9-Glut1 Wild-type (WT) WT + AAV9-Glut1

b

N.S.

N.S.

*** *** *

*

*

N.S.

c

1 2 3 4 8 2 0

0

2

4

6

8

1 0

A g e (w e e k s )

Re

lati

ve

RN

A e

xp

res

sio

n (

Glu

t1

-ac

tin

)

W T

M u t

Heart

* *

* d

0 .0

0 .6

1 .2

1 .8

4 0

8 0

1 2 0

1 6 0

Re

lati

ve

RN

A e

xp

res

sio

n (

Glu

t1

-ac

tin

)

anti-eGFP anti-Glut1 Merge

a

*

*

anti-GFAP

22mm

0 .0

0 .5

1 .0

1 .5

2 .0

Re

lati

ve

RN

A e

xp

res

sio

n (

Glu

t1

-ac

tin

)

Kidney

** N.S.

**

Muscle

*** ***

*** e f

Mut + vehicle Mut + AAV9-Glut1 Wild-type

Liver

*** N.S.

***

g

0 .0

0 .5

1 .0

1 .5

3 0

4 0

5 0

6 0

Re

lati

ve

RN

A e

xp

res

sio

n (

Glu

t1

-ac

tin

)

0 . 0

0 .5

1 .0

1 .5

Re

lati

ve

RN

A e

xp

res

sio

n (

Glu

t1

-ac

tin

)

Supplementary Figure. 6. Assessing the delivery and effects of AAV9-Glut1 on Glut1 DS model mice. (a) Immuno-histochemistry of brain (hippocampus) sections of mice administered AAV9-eGFP showing transduction of the Glut1-positive cells of the cerebral microvasculature by the vector (arrows). Note – asterisks denote astrocytes. (b) Graphical representation of improved motor performance on the rotarod following administration of the AAV9-Glut1 vector through the intra-cerebral ventricles of PND3 Glut1 DS model mice. Systemic administration of the vector into WT mice did not improve performance above that observed in naïve WT mice. ***, P < 0.001, t test, N ≥ 7 mice in each cohort. (c) Longitudinal assessment of Glut1 mRNA expression in brain tissue of Glut1 DS mutants and control, WT littermates. Expression peaked at 3 weeks of age in both cohorts, leveling off at ~8 weeks. A significant difference between mutants and controls became apparent at 2 weeks. *, ***, P < 0.05 and P < 0.001 respectively, t test, N ≥ 3 mice of each genotype. (d – g) Glut1 repletion in mutants at PND3 significantly raises Glut1 RNA expression in diverse organs. *, **, ***, P < 0.05, P < 0.01 and P < 0.001 respectively, one-way ANOVA, N ≥ 3 mice in each cohort.

Supplementary Figure 7. Effects of early Glut1 repletion on the hypoglycorrhachia, micrencephaly and epileptiform activity of Glut1 DS model mice. Longitudinal assessment of (a) blood glucose, (b) CSF glucose and (c) the ratio of CSC:blood glucose in naïve (untreated) mutants and WT littermates demonstrates unchanged blood glucose values. In contrast, significant differences in CSF glucose values and CSF:blood glucose ratios become evident as early as 1 week of life. Moreover, in mutants both values declined significantly from a peak recorded at 3 weeks of age. ***, P < 0.001, t test, N ≥ 7 mice of each genotype. (d) Body weight of Glut1 DS mutants is no different from that of WT, control littermates between 1 and 20 weeks of age. However, diminished brain size, a measure of micrencephaly becomes evident in mutants (e) no later than PND14. *, **, ***, P < 0.05, P < 0.01, P < 0.001 respectively, t test, N ≥ 5 mice of each genotype for (D) and (E). AAV9-Glut1 delivered early into the intra-cerebral ventricles is also effective in (f, g) mitigating hypoglycorrhachia and (h) restoring normal brain size to Glut1 DS model mice. **, ***, P < 0.01 and P < 0.001 respectively, one-way ANOVA, N ≥ 8 mice in each group. Results of EEG analysis depicting (i) representative brain-wave activity in the three cohorts of mice and (j) a quantification of epileptic seizure-like events. *, P < 0.05, one-way ANOVA, N ≥ 3 mice in each group. Note persistence of spontaneous, generalized seizures (arrow) in the vehicle-treated animal.

0 1 2 3 4 5 6

0

2 0

4 0

6 0

8 0

1 0 2 0

A g e (w e e k s )

CS

F g

luc

os

e (

mg

/dL

)

a b c

d e

0

1 0

2 0

3 0

Bo

dy

we

igh

t (g

)

N.S.

N.S.

N.S.

N.S. N.S.

N.S.

WT Mut

N.S.

*** *** * ** ***

0 .0

0 .1

0 .2

0 .3

0 .4

0 .5

Bra

in w

eig

ht

(g)

1 0

1 5

2 0

2 5

3 0

3 5

0 .1

0 .2

0 .3

0 .4

0 .5

Bo

dy

we

igh

t (g

)

Bra

in w

eig

ht (g

)

0

5 0

1 0 0

1 5 0

0

2 0

4 0

6 0

8 0

1 0 0

Blo

od

glu

co

se

(m

g/d

L) C

SF

glu

co

se

(mg

/dL

)

N.S.

N.S.

***

*** N.S.

***

*** N.S.

*

N.S. f g h

N.S.

* N.S.

*

Mut + AAV9-eGFP (Facial vein) Mut + AAV9-Glut1 (Facial vein)

Wild-type Mut + AAV9-Glut1 (ICV)

Mut + vehicle

N.S.

0 .0

0 .3

0 .6

0 .9

1 .2

CS

F/B

loo

d G

luc

os

e R

ati

o

0 . 0

0 .2

0 .4

0 .6

0 .8

Nu

mb

er

of

se

izu

res

/ho

ur

*

Mut + vehicle Mut + AAV9-Glut1 Wild-type

i j

0

2 0

4 0

6 0

8 0

1 0 0

Blo

od

glu

co

se

(m

g/d

L)

N.S.

WT Mut

N.S. N.S. N.S. N.S. N.S. N.S.

*** ***

*** ***

*** ***

Mut WT

0 1 2 3 4 5 6

0 .0

0 .2

0 .4

0 .6

0 .8

1 .0

1 0 2 0

A g e (w e e k s )CS

F/B

loo

d G

luc

os

e R

ati

o

*** ***

*** ***

*** ***

Mut WT

N.S.

Mut + v

ehic

le (

2 m

o)

Mut + A

AV

9-G

lut1

(2m

o)

Mut + A

AV

9-G

lut1

(4m

o)

Mut + A

AV

9-G

lut1

(8m

o)

0 .0

0 .5

1 .0

1 .5

2 .0

2 .5

Re

lati

ve

RN

A e

xp

res

sio

n

(Glu

t1

-ac

tin

)

b

N.S.

***

Wild-type a

Mut + vehicle Se

pto

-str

iatu

m

Hip

po

cam

pu

s Th

alam

us

Cer

ebel

lum

Li

ver

Hea

rt

Kid

ney

M

usc

le

Smal

l In

test

ine

Larg

e In

test

ine

Mut + Glut1-AAV9

100mm

100mm

100mm

100mm

100mm

100mm

100mm

100mm

100mm

100mm

Supplementary Figure 8. Early administration of AAV9-Glut1 allows for sustained expression of the Glut1 gene and does not trigger major pathology in treated mice. (a) Hematoxylin/eosin histochemistry of the major organs of the body at 5 months of age depicts normal cellular morphology in the majority of the tissues of mutants treated with AAV9-Glut1. A hint of steatosis within the liver tissue is detected; note somewhat lighter staining of tissue (asterisk) surrounding central vein in the sample from the mutant treated with the Glut1 virus. (b) QPCR analysis of Glut1 transcripts in brain tissue reveals sustained expression of the Glut1 transgene as late as 8 months of age. Note significant increase in expression relative to that in the vehicle-treated mutant. ***, P < 0.001, one-way ANOVA, N ≥ 3 mice in each cohort.

a

N.S.

Mut + v

ehic

le

Mut (P

ND

3)

+ A

AV

9-G

lut1

Mut (2

wk)

+ A

AV

9-G

lut1

0

5 0

1 0 0

1 5 0B

loo

d g

luc

os

e (

mg

/dL

)

Supplementary Figure 9. Blood glucose levels in mutants provided food ad libitum. Blood glucose levels remain unaltered in non-fasted 5-month old mutants administered AAV9-Glut1. P > 0.05, one-way ANOVA, N = 3 – 8 mice in each cohort.

Full-sized western blots

Vegfr2

-actin

Glut1

Fig. 3b

40kDa 50kDa

40kDa 50kDa

30kDa

SMN

Glut1

Fig. 3e

40kDa 30kDa

50kDa

40kDa 30kDa

50kDa

Fig. 4d – upper panel

Glut1

SMN

40kDa 30kDa

50kDa

40kDa 30kDa

50kDa

SMN

Glut1

Fig. 4d – lower panel

40kDa 30kDa

50kDa

40kDa 30kDa

50kDa

SMN

Glut1

Fig. 6c

50kDa 40kDa

80kDa 120kDa

30kDa

50kDa 40kDa

80kDa 120kDa

30kDa

120kDa 220kDa

Supplementary Figure 10. Images of full-sized western blots.